Method of osteogenic differentiation in microfluidic tissue culture systems

ABSTRACT

Microfluidic “organ-on-a-chip” devices have been developed with the aim to replicate human tissues in vitro. However, there is no option to quantitatively monitor biological processes that take place within the chip, over time. Destructive methods in order to analyze, tissue formation, gene expression, protein secretion etc. require the harvest of the “tissue” at a certain time point. Described herein are methods and compositions for non-destructive molecular imaging methods and systems in order to quantitatively monitor specific biological processes, over time, within the chip, without the need to harvest.

FIELD OF THE INVENTION

Described herein are methods and compositions related to imaging of stemcells and cells undergoing differentiation without sample manipulation.

BACKGROUND

Bones consisting of mineralized bone tissue also consists of bonemarrow, nerves and blood vessels. Development and homeostasis of bonerelies heavily on communication between cells in the tissues asregulated by the bone environment. Bone is an active tissue maintainedby bone cells such as osteoblasts that form bone and osteoclasts thatresorb bone, and it is now understood that mesenchymal stem cells (MSCs)can differentiate to various skeletal cells including osteoblasts,chondrocytes, fibroblasts, adipocytes, tenocytes, nucleus pulposus cellsand more. Additionally, within the collagen and mineral matrixosteocytes are also embedded and respond to the bone environment. Thebalance between these cells is necessary to maintain bone function.Studying bone is a challenging field due to microarchitecture definingthe bone environment, which involve the intricately dense structuralcomposition of the bone morphology. Unlike other tissues that can beprocessed and prepared for experiments, including cultured cell lines,working with bone is difficult. Studying intracellular dynamics of thebone cells embedded within the mineralized tissue has proven to be achallenging task.

Compounding these challenges related to underlying properties of thecellular material, imaging cells at subcellular level within the boneenvironment is very difficult. Paraffin tissue slices are a longstanding conventional approach to evaluate microarchitecture and bonemorphology. However, sample manipulation leads to changes in biochemicalproperties of antigenicity and mineral structure. Newer strategies toimage cells within the bone such as MRI, Micro-CT or Ultrasound canimage bone structure and recently cells, however these techniques arelimited by their low resolution at the cellular level given thesurrounding physiological environment.

Recent “organ-on-a-chip” technologies represent new and excitingopportunities for bone research. These devices include a microfluidiccell culture apparatus that is a more physiologically relevant in vitromodel than cells cultured in dishes. Importantly, providing forcontinuously perfused chambers inhabited by living cells arranged tosimulate tissue- and organ-level physiology allow for the culturing ofbone cells in a format mirroring their physiological environment. Bystudying bone cell function and response in this manner, a 3Denvironment can reveal completely different cellular dynamics comparedto 2D cultures. The availability of cellular tissue material in thisformat further provides new avenues for apply imaging approaches of bonemicroarchitecture to identify features previously unavailable in tissuecultures or at insufficient resolution in vivo. Real time imaging of thebone marrow niche within bone and fluorescent imaging of cells withinthe bone marrow niche has reportedly been achieved. Recent advancementsin imaging techniques allows for the identification of osteocytesembedded in the bone matrix. However, determining the localization ofcell types and protein expression dynamics of single cells within thebone is still very difficult. And more research is needed to identifyintracellular protein activities of the cell bodies embedded withinmineralized matrix.

Described herein is the use of non-destructive molecular imaging methodsand systems in order to quantitatively monitor specific biologicalprocesses, over time, within the chip, without the need to harvest thetissue. Such methods can provide valuable data on developing tissues andtheir response to pharmaceutical, chemical and environmental agents.

SUMMARY OF THE INVENTION

Described herein is a method of detecting cellular mineralization in amicrofluidic device including providing a microfluidic device includingmesenchymal stem cells (MSCs), osteoblasts and/or osteocytes, adding oneor more labeling agents to the microfluidic device, and detecting thelabeling agent, wherein the labeling agent is capable of binding tocellular mineralization. In other embodiments, the microfluidic devicefurther includes one or more channels for loading of a control sample.In other embodiments, the one or more labeling agents comprisebisphosphonate imaging agents. In other embodiments, the bisphosphonateimaging agent includes a pamidronate backbone with a fluorescent label.In other embodiments, the one or more labeling agents comprise aradiolabel. In other embodiments, the radiolabel includes technetium-99m([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP),and/or 68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1O-bis(carboxymethyl)I,4,7,I0-tetraazacyclododec-1-yl)acetic acid (BPAMD)([68Ga]BPAMD). In other embodiments, detecting the labeling agentincludes Micro CT, Micro SPECT, and/or PET imaging. In otherembodiments, detecting the labeling agent further includes comparison ofthe quantity of detected labeling agent with one or more controlsamples. In other embodiments, further culturing of MSCs, osteoblasts,osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells,and/or nucleus pulposus cells in the microfluidic device. In otherembodiments, the method includes further detection of the labelingagent.

Also described herein is a method of detecting secreted extracellularmacromolecules in a microfluidic device including providing amicrofluidic device including stem cells, applying one or more pulsesequences to the microfluidic device; and, detecting the pulse sequencesignal intensity, wherein the pulse sequence signal intensity is capableof measuring one or more macromolecules secreted by the stem cells. Inother embodiments, the stem cells are mesenchymal stem cells (MSCs). Inother embodiments, the stem cells are pluripotent stem cells (pSCs). Inother embodiments, the stem cells are induced pluripotent stem cells(iPSCs). In other embodiments, detecting the pulse sequence signalintensity includes chemical exchange saturation transfer (CEST), pHmeasurement of T1 rho, magnetization transfer contrast (MTC), and/ormagnetization exchange (MEX). In other embodiments, CEST detects aquantity of glycosaminoglycans (GAGs). In other embodiments, pHmeasurement of T1 rho detects a quantity of GAGs. In other embodiments,MTC detects a quantity of collagen. In other embodiments, MEX detects aquantity of collagen and/or osteoid. In other embodiments, themicrofluidic device further includes one or more channels for loading ofa control sample. In other embodiments, detecting the pulse sequencesignal intensity further includes comparison of the quantity of detectedpulse sequence signal intensity with one or more control samples. Inother embodiments, the method includes further culturing of stem cellsin the microfluidic device. In other embodiments, the method includesfurther detection of pulse sequence signal intensity.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Organ-on-chip dimensions and setting of the flow system. Theflow was set to 30 μl/h and the media in the reservoirs was replaced orrefilled twice a week.

FIG. 2. Cell survival and proliferation on the organ-on-chip. FIG. 2A.Micrographs of the cells on the chip grown for 3 weeks in osteogenicconditions. FIG. 2B. bioluminescent imaging (BLI) images taken on Day 0.FIG. 2C. Quantitative analysis BLI that was done twice a week for 3weeks. Bars indicate standard deviation, n=5, *p<0.05; ***P<0.001.

FIG. 3. Osteogenic differentiation at Week 3 measured with two probes(OsteoSense and BoneTag) using two different imaging systems:fluorescent imaging system (IVIS, Perkin Elmer) and Near Infraredimaging system (Odyssey® CLx, Li-Cor). FIG. 3A. FLI images of chipsincubated with BoneTag and in FIG. 3B the labeling was quantified FIG.3C. using fluorescent imaging IVIS Live staining where live cells artestained with FITC (green) dye and OsteoSense is depicted in red andimaged using confocal microscopy, 10× magnification. In FIG. 3D,labeling was quantified, bars indicate standard deviation, n=5, *p<0.05;***P<0.001.

FIG. 4. Immunostaining of osteogenic differentiation of MSC-BMP2 inOrgan-on-chip. The chips were sectioned using vibratome across thechannels. The sections were stained using with immunofluorescentstaining against the osteogenic markers osteocalcin and bonesialoprotein (BSP), and imaged using confocal microscopy.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Allen et al., Remington: The Science and Practice of Pharmacy22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al.,Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology 3^(rd) ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006);Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7^(th) ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton,Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (Nov.28, 2012); and Green and Sambrook, Molecular Cloning: A LaboratoryManual 4^(th) ed., Cold Spring Harbor Laboratory Press (Cold SpringHarbor, N.Y. 2012), provide one skilled in the art with a general guideto many of the terms used in the present application. For references onhow to prepare antibodies, see Greenfield, Antibodies A LaboratoryManual 2^(nd) ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y.,2013); Köhler and Milstein, Derivation of specific antibody-producingtissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976July, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat.No. 5,585,089 (1996 December); and Riechmann et al., Reshaping humanantibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

In recent years, microfluidic devices have been developed with the aimto replicate human tissues in vitro. These systems, also calledmicrofluidic chips or “organ-on-a-chip”, have the potential to serve asan alternative for animal models that are used to test pharmaceutical,chemical and environmental agents. The microfluidic chips are attractivefor biomedical research and drug discovery due to low cost and ethicalconsiderations compared to animal models. A variety of examples aredescribed in Bhatia and Ingber, “Microfluidic organs-on-chips.” NatBiotechnol. 2014 August; 32(8):760-72, which is fully incorporated byreference herein.

An important caveat of the “‘chips” is that currently there is no optionto quantitatively monitor biological processes that take place withinthe chip, over time. To date, researchers are using destructive methodsin order to analyze tissue formation, gene expression, protein secretionetc. These methods include histology, immunofluorescence or PCR andrequire the harvest of the “tissue” at a certain time point. The use ofnon-destructive molecular imaging methods and systems in order toquantitatively monitor specific biological processes, over time, withinthe chip, without the need to harvest the tissue would be a significantimprovement in the field. Such methods can provide valuable data ondeveloping tissues and their response to pharmaceutical, chemical andenvironmental agents.

Mesenchymal stem cells (MSCs) can differentiate to various skeletalcells including osteoblasts, chondrocytes, fibroblasts, adipocytes,tenocytes, nucleus pulposus cells and more. In situ imaging, both innon-living and living specimens, have provided new insights, but for theabove described reasons, quantitative experimental data requiresdestructive processing that may introduce bias, and lack temporal andspatial resolution. In this regard, microfluidic organ-on-a-chip coupledwith non-destructive labeling and imaging techniques may allow precisecapture of MSC, osteoblast and osteocyte cell populations in micro andultrastructure in 2D and 3D. Live cell imaging techniques which are ableto track structural morphology and cellular differentiation in bothspace and time combined with the latest biochemical assays andmicrofluidic imaging techniques can provide further insight on thebiological function of MSC, MSCs, osteoblasts, osteocytes, chondrocytes,tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposuscells.

Existing techniques for imaging of cells in skeletal and other tissueshas proved challenging due to the need to develop methodologies forsectioning specimens, labeling or imaging of specimens or to developprotocols for decalcifying specimens to enable conventional sectioningand imaging techniques to be used. Current imaging approaches relymainly on histological stains combined with conventional lightmicroscopy. Confocal imaging approaches allows for three-dimensional(3D) imaging in situ within the bone environment. In contrast toinherently two-dimensional (2D) imaging techniques such as lightmicroscopy, confocal microscopy stacks optical sections at differentfocal planes to generate a three-dimensional (3D) representation of thesample. Endogenous (auto)fluorescence of the bone tissue can be used toprovide contrast for confocal microscopy measurements. More often,various fluorescent staining agents are used in conjunction with modernconfocal laser scanning microscopy (CLSM), such as rhodamine andfluorescein, which can be incubated with undecalcified bone sections.More specific staining agents, such as fluorescein isothiocyanate(FITC)-conjugated phalloidin and DAPI, label the actin skeleton and/orDNA of cell nuclei in such a way that the components cells can bedirectly imaged and separately displayed in 3D

However, a major drawback with CLSM is the limited maximum focal planedepth of around 100-150 Additionally, CLSM is tainted with imageartifacts, such as signal attenuation with increasing focal plane depthor aberrations due to refractive index mismatch. Such artifacts areabsent in (conventional) X-ray absorption-based computed tomography(CT). Micro-computed CT (μCT) and 3D morphometric measures to quantifytrabecular microarchitecture have laid the foundations for μCT to becomea standard for bone morphometry. In bone research, the standardapplication of desktop μCT systems with typical voxel sizes in the orderof 5-100 μm is a core approach for quantitative characterization ofwhole bone geometry. Synchrotron radiation-based CT allows for imagingof bone microstructure, canal networks, as well as study of populationssuch as osteocytes within bone. Most recently, optimized imagingprotocol for SR CT provides spatial resolution closer to the diffractionlimit of visible light at a few hundred nanometers. The recentavailability of desktop μCT scanners with voxel sizes below 1 μm allowfor new opportunities for imaging.

Over the past two decades or so, technologies for imaging of livingcells using light and confocal microscopy have advanced at a rapid rate.Coupled with enhanced green fluorescent proteins (GFPs) and a seeminglylimitless array of fluorescent imaging probes has made it possible toimage almost any intracellular or extracellular structure or protein inliving cells and tissues. A large selection of fluorescent probes andreagents are commercially available to the researcher for investigatingbiological events in living cells, including fluorescent antibodies,kits for fluorescently labeling proteins of interest, dyes for cell andnuclear tracking, probes for labeling of membranes and organelles,fluorescence reagents for determining cell viability, probes forassessing pH and ion flux and probes for monitoring enzyme activity,etc. In addition, a variety of GFP-derived fluorescent protein vectorsare available that can either be used as reporter constructs or togenerate fusion constructs with a protein of interest. These enable thelive monitoring of gene expression and protein localization in vivo, andin real time.

The traditional approach of collecting “static” images of fixed or postmortem cells and tissues provides a snapshot view of events at a singlefixed point in time. However, this inherently overlooks the dynamicaspects of the biology being examined. In contrast, live cell imagingenables the visualization of temporal changes in living specimens andcan reveal novel aspects of the biology that may not otherwise have beenappreciated. Additionally, the datasets generated from time-lapseimaging are information rich and can be interrogated quantitatively toenable measurement of cellular, subcellular and tissue dynamic events asa function of time

Although these approaches are leading to exciting discoveries that areadvancing our understanding of biological systems, there are severallimitations that need to be acknowledged. Firstly, fluorescent probesmay perturb or alter the biology being examined. Validation studies areneeded to make sure that the fusion protein still functions similarly tothe wild type form. It is also advantageous to confirm findings withmore than one type of imaging probe if possible. For example, a GFPfusion protein can be used for in vivo localization of a specificprotein and key data can be confirmed using a fluorescence-conjugatedantibody against the same protein. When developing live cell imagingprotocols, there is always a compromise between obtaining a high enoughsignal-to-noise ratio to enable quantitative measurements and to obtainsufficient image resolution, while at the same time avoiding phototoxiceffects to the cells. Therefore, to ensure cell viability, theresearcher may have to accept a lower image quality and resolution thanwould be acceptable for equivalent images of fixed specimens.Nevertheless, technologies such as multiphoton fluorescence microscopycan increase the depth of tissue penetration for live cell imagingapplications and reduce phototoxicity by using a longer wavelength lightto excite fluorophores. These instruments are becoming more widely usedfor live imaging applications due to their advantages over conventionalwidefield and confocal microscopy systems.

Recently, live cell imaging approaches have been applied to the study ofMSCs, osteoblasts and osteocytes. Organ cultures of neonatal calvariafrom mice have provided a useful model for imaging the dynamicproperties of osteocytes. Another way in which this model can be usedfor imaging osteocyte dynamics is by using long term cultures of MSCsand osteoblasts. These cells differentiate when cultured undermineralizing conditions to form mineralized nodules in which thetransition to the osteocyte-like phenotype can be monitored byfluorescent labeling or radiolabeling. To gain maximum information,imaging of these can be combined with other fluorescent probes, such asalizarin red to monitor mineral deposition. Live cell imaging studies asapplied to investigating osteocyte biology are still in their infancy.In addition to revealing the dynamic properties of MSCs, osteoblasts andosteocytes, identifying the underlying intracellular signaling pathways,such as calcium oscillations, monitoring the temporal integration ofosteocyte differentiation and mineralization, live imaging studies haveconsiderable potential to address many as yet unresolved questions inosteocyte biology.

Most importantly, biochemical data characterizing the precise role ofMSCs, osteoblasts and osteocytes in bone remodeling remains severelylimited. A number of in vivo models have been developed to study theirfunction. Existing technologies typically harvest large osteocytepopulations and employ technologies which provide a comprehensiveassessment of a large number of genes which are both up-regulated anddown-regulated in response mechanical stimulation. For example, tocomprehensively assess osteocyte gene expression in a mouse model forload induced bone adaptation, current state-of-the-art approachesextract large populations of osteocytes from loaded bone and performmicro-array-analysis to quantify the expression levels of tens ofthousands of different genes. Global gene expression assays derived fromin vivo models for bone adaptation have identified a number of candidategenes and revealed potential load regulated pathways. Nevertheless,there are significant limitations when interpreting these data. Theharvesting and analysis of large populations of osteocytes reports geneexpression averaged over tens of thousands of cells, each of whichreside in different micro-environments characterized by different levelsof mechanical strain and local osteoblastic/osteoclastic activity. It istherefore possible that key genes and networks are being concealed.Emerging studies investigate local regulation of gene expression inosteocytes by comparing 2D histology sections from loaded bone stainedfor specific molecular targets (sclerostin) with micro finite element(μFE) models. Whilst informative, these approaches are still very muchqualitative and only permit the analysis of one specific moleculartarget at a time.

Addressing these limitations are microfluidic imaging approaches whichallow for spatial and temporal mapping in three dimensions andquantitative measurement of gene expression cells in an organized“organ-on-a-chip” niche. Examples of a “microfluidic imaging” approachcan be briefly described by the following workflow: bone formationand/or resorption are spatially mapped and quantified in technologiessuch as in vivo μCT and 3D image registration techniques; labeling(e.g., fluorescence, radio labeling) or other techniques, (e.g.,chemical exchange saturation transfer (CEST), pH measurement T1rho,magnetization transfer contrast, magnetization exchange or othertechnologies. The vast amount of data generated using these approachescan be used to build, feed and validate computational models of variousskeletal and other tissues, which incorporate all of the differentlength scales, from the organ-level to the cellular-level. Furtherexamples include those described in Trussel et al., “Toward mechanicalsystems biology in bone.” Ann Biomed Eng. 2012 November; 40(11):2475-87.

Described herein is a method of detecting properties of one of morecells in a microfluidic device. In other embodiments, the microfluidicdevice includes mesenchymal stem cells (MSCs), osteoblasts and/orosteocytes. In other embodiments, the microfluidic device includescartilage, tendon/ligament, nucleus pulposus, annulus fibrosus,chondrocytes, tenocytes, fibroblasts, and/or notochordal cells amongothers. It is emphasized that the described methods and techniques findwide applicability to biological tissues. In other embodiments, themicrofluidic device includes stem cells. In other embodiments, the stemcells are mesenchymal stem cells (MSCs). In other embodiments, the stemcells are induced pluripotent stem cells (iPSCs). In other embodiments,the microfluidic device further includes one or more channels forloading of a control sample. In various embodiments, the properties arebiochemical properties of the one or more cells in a microfluidicdevice.

In various embodiments, the method includes providing a microfluidicdevice, adding one or more labeling agents to the microfluidic device,and detecting the labeling agent, wherein the labeling agent is capableof binding to one or more biochemical properties of one or more cells inthe microfluidic device. In other embodiments, one or more labelingagents comprise bisphosphonate imaging agents. In other embodiments, thebisphosphonate imaging agent includes a pamidronate backbone with afluorescent label. In other embodiments, the one or more labeling agentscomprise a radiolabel. In other embodiments, the radiolabel includestechnetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate(Tc-MDP), and/or 68Ga-Labeled(4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1O-bis(carboxymethyl)-I,4,7,I0-tetraazacyclododec-1-yl)acetic acid(BPAMD) ([68Ga]BPAMD). In other embodiments, detecting the labelingagent includes Micro CT, Micro SPECT, and/or PET imaging. In otherembodiments, detecting the labeling agent further includes comparison ofthe quantity of detected labeling agent with one or more controlsamples. In other embodiments, the method includes further culturing ofMSCs, osteoblasts and/or osteocytes in the microfluidic device. In otherembodiments, the method includes further detection of the labelingagent.

In various embodiments, the method includes applying one or more pulsesequences to the microfluidic device, and detecting the pulse sequencesignal intensity, wherein the pulse sequence signal intensity is capableof measuring one or more biochemical properties. In other embodiments,detecting the pulse sequence signal intensity includes chemical exchangesaturation transfer (CEST), pH measurement of T1 rho, magnetizationtransfer contrast (MTC), and/or magnetization exchange (MEX). In otherembodiments, CEST detects a quantity of glycosaminoglycans (GAGs). Inother embodiments, pH measurement of T1 rho detects a quantity of GAGs.In other embodiments, MTC detects a quantity of collagen. In otherembodiments, MEX detects a quantity of collagen and/or osteoid. In otherembodiments, the microfluidic device further includes one or morechannels for loading of a control sample. In other embodiments,detecting the pulse sequence signal intensity further includescomparison of the quantity of detected labeling agent with one or morecontrol samples. In other embodiments, the method includes furtherculturing of stem cells in the microfluidic device. In otherembodiments, the method includes further culturing of cartilage,tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes,tenocytes, fibroblasts, and/or notochordal cells among others. In otherembodiments, the method includes further detection of pulse sequencesignal intensity. In various embodiments, the method includes detectingcellular mineralization. In other embodiments, the method includesdetecting secreted extracellular macromolecules. In various embodiments,the method includes detecting cellular survival, differentiation and/orproliferation.

Described herein is a method of detecting cellular mineralization in amicrofluidic device including providing a microfluidic device includingmesenchymal stem cells (MSCs), osteoblasts and/or osteocytes, adding oneor more labeling agents to the microfluidic device, and detecting thelabeling agent, wherein the labeling agent is capable of binding tocellular mineralization. In other embodiments, the microfluidic devicefurther includes one or more channels for loading of a control sample.In other embodiments, the one or more labeling agents comprisebisphosphonate imaging agents. In other embodiments, the bisphosphonateimaging agent includes a pamidronate backbone with a fluorescent label.In other embodiments, the one or more labeling agents comprise aradiolabel. In other embodiments, the radiolabel includes technetium-99m([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP),and/or 68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1O-bis(carboxymethyl)-I,4,7,I0-tetraazacyclododec-1-yl)acetic acid(BPAMD) ([68Ga]BPAMD). In other embodiments, detecting the labelingagent includes Micro CT, Micro SPECT, and/or PET imaging. In otherembodiments, detecting the labeling agent further includes comparison ofthe quantity of detected labeling agent with one or more controlsamples. In other embodiments, the method includes further culturing ofMSCs, osteoblasts and/or osteocytes in the microfluidic device. In otherembodiments, the method includes further culturing cartilage,tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes,tenocytes, fibroblasts, and/or notochordal cells in the microfluidicdevice. In other embodiments, the method includes further detection ofthe labeling agent.

Also described herein is method of detecting secreted extracellularmacromolecules in a microfluidic device including providing amicrofluidic device including stem cells, applying one or more pulsesequences to the microfluidic device, and detecting the pulse sequencesignal intensity, wherein the pulse sequence signal intensity is capableof measuring one or more macromolecules secreted by the stem cells. Inother embodiments, the stem cells are mesenchymal stem cells (MSCs). Inother embodiments, the stem cells are induced pluripotent stem cells(iPSCs). In other embodiments, detecting the pulse sequence signalintensity includes chemical exchange saturation transfer (CEST), pHmeasurement of T1 rho, magnetization transfer contrast (MTC), and/ormagnetization exchange (MEX). In other embodiments, CEST detects aquantity of glycosaminoglycans (GAGs). In other embodiments, pHmeasurement of T1 rho detects a quantity of GAGs. In other embodiments,MTC detects a quantity of collagen. In other embodiments, MEX detects aquantity of collagen and/or osteoid. In other embodiments, themicrofluidic device further includes one or more channels for loading ofa control sample. In other embodiments, detecting the pulse sequencesignal intensity further includes comparison of the quantity of detectedpulse sequence signal intensity with one or more control samples. Inother embodiments, the method includes further culturing of stem cellsin the microfluidic device. In other embodiments, the method includesfurther culturing cartilage, tendon/ligament, nucleus pulposus, annulusfibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cellsin the microfluidic device. In other embodiments, the method includesfurther detection of pulse sequence signal intensity.

Example 1 Micro Imaging for Non Invasive Monitoring of Stem Cell-InducedMineralization

Mesenchymal stem cells (MSCs) can differentiate to various skeletalcells including osteoblasts. A common assay of MSC differentiation toosteogenic cells includes measurements of mineralization within theculture. Several methods can be used to monitor mineralization over timein chips

Example 2 Labeling Agents for Stem Cell-Induced Mineralization

Fluorescence imaging—bisphosphonate imaging probes such as OsteoSense™(Perkin Elmer) can be added to the chip at different time points, washedand then the chip is imaged in an optical scanner. Hydroxyapatite (HA)is a mineral form of calcium apatite and is the major mineral product ofosteoblasts. Therefore, HA levels are a good biomarker for osteoblastactivity. In addition, abnormal accumulation of HA can be indicative ofa disease state. OsteoSense™ imaging agents bind with high affinity toHA. Since hydroxyapatite (HA) is known to bind pyrophosphonates andphosphonates as well as synthetic bisphosphonates with high affinity,OsteoSense™ agents were designed as bisphosphonate imaging agents. Theseprobes consist of a pamidronate backbone functionalized withnear-infrared fluorophore off the amino terminus of the R2 side chain.Specifically, OsteoSense™ imaging agents can be used to image areas ofmicrocalcifications, bone remodeling and enables imaging of bone growthand resorption. The bisphosphonate probe attaches to microcalcifications and the fluorescent readout provides quantification ofmineralization.

Example 3 Other Labeling Agents for Stem Cell-Induced Mineralization

Bisphosphonates (BPs; also known as diphosphonates), such as methylenediphosphonate (MDP) and zoledronic acid, can be labeled withtechnetium-99m ([99mTc]-BPs) for use in bone scintigraphy as has beenused to detect osteoporosis and other skeletal-related events (SREs).These chemicals bind hydroxyapatite, which allow for imaging ofbisphosphonates as described above. [18F]-Fluoride is another nuclidethat is commonly used for bone imaging, and positron emission tomography(PET) and is believed to be superior to [99mTc]-BPs for the diagnosis ofSREs.

Micro SPECT/PET imaging-99mTc-Methyl diphosphonate (Tc-MDP) can be addedto the chip at different time points, washed and then the chip is imagedusing a micro SPECT scanner. Alternative probes are [‘8F)-Fluoride or68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1O-bis(carboxymethyl)-I,4,7,I0-tetraazacyclododec-1-yl)acetic acid(BPAMD) [68Ga]BPAMD that can be imaged using a micro PET scanner. Theseprobes also attach to mineralization foci and the uptake readouts canprovide quantitative data of mineralization.

Example 4 Micro Imaging for Non Invasive Monitoring of Stem Cell-InducedMineralization

Micro CT—high-resolution micro CT scanners can detect mineral particlesas small as 500 nm. A non-destructive scan of the chip can provide anaccurate measurement of mineralization generated by the developingtissues.

Example 5 Micro Imaging for Non Invasive Monitoring of ExtracellularMacromolecules Secreted by Stem Cells

Different types of stem cells including MSCs and induced pluripotentstem cells (iPSCs) have been shown to differentiate to joint tissuecells such as osteoblasts, osteocytes, chondrocytes, tenocytes,fibroblasts, notochordal cells, and/or nucleus pulposus cells cells.While differentiating, the cells secret characteristic extracellularmolecules such as aggrecan, glycosaminoglycans (GAGs), collagens andmore.

A way to monitor the secretion of these molecules in a chip will includethe use of micro MRI using different pulse sequences, including but notlimited to: chemical exchange saturation transfer (CEST)—GAGsmeasurement; pH measurement T1 rho—GAGs measurement, magnetizationtransfer contrast (MTC)—collagen measurement, magnetization exchange(MEX)—collagen and osteoid measurement.

Chemical exchange saturation transfer (CEST) also provides the abilityto analyze the GAG content in cartilage. The most common method foracquisition of a CEST data set is to acquire multiple image data setswith presaturation at different offset frequencies around the waterresonance and one reference data set without saturation or withsaturation at a very large offset frequency. The normalized signal as afunction of the presaturation offset (termed the z-spectrum) can then beused to determine and quantify CEST effects, which are asymmetric withrespect to the water resonance (ie, a CEST effect appears either up- ordown-field from water and therefore can be extracted from the z-spectrumvia analysis of its asymmetry with respect to the water resonance).

Chemical exchange saturation transfer (CEST) is a magnetic resonanceimaging (MM) contrast enhancement technique that enables indirectdetection of metabolites with exchangeable protons. Endogenousmetabolites with exchangeable protons including many endogenous proteinswith amide protons, glycosaminoglycans (GAG), glycogen, myo-inositol(MI), glutamate (Glu), creatine (Cr) and several others have beenidentified as potential in vivo endogenous CEST agents. These endogenousCEST agents can be exploited as non-invasive and non-ionizing biomarkersof disease diagnosis and treatment monitoring.

Magnetization Transfer Contrast (MTC) MRI is an imaging method thatevolved from NMR spectroscopy. In tissue imaging, MTC relies upon theinteraction of less mobile protons associated with macromolecules suchas proteins and their interactions with protons freely associated withwater. The premise is that in a system where molecules move and exchangeposition, whether it be a change in spatial position in asymmetricalmolecules or an exchangeable proton between a molecule and water, themagnetization state will also move and be transferred.

A two pool model can be utilized to illustrate the theory behind MTCMRI. Conventional MRI detects only the free water pool while themacromolecular pool remains mostly undetected. Both the macromolecularand free water pools are centered around the same frequency but themacromolecular pool is shallower and wider. Saturation is achieved byapplying an off-resonance radio frequency (RF) pulse specific to a peakin the macromolecular pool before excitation at the center frequency.The RF pulse saturates the signal from the section leading to ideally nosignal at the off-resonance frequency. Since both pools interact thissaturation is transferred to the free water pool. While it is notpossible to detect the changes in the macromolecular pool directly, itcan be assumed that the loss in signal intensity of the free water poolcorresponds to the changes in the macromolecular pool.

Ideally, an increase is preferable to a decrease in signal intensitysince it is easier to visualize changes in brightness over changes indarkness. To achieve this type of image, a Magnetization Transfer Ratio(MTR) is calculated using a base image without saturation to measure therelative loss of signal intensity in a pixel by pixel basis:MTR=Nonsaturated−Saturated/Nonsaturated. MTC is very similar in functionto CEST. CEST focuses on a limited part of magnetization transfer bylinking it to chemical exchange systems.

Quantitative magnetization transfer (qMT) imaging is MR technique whichutilizes a two-pool model of magnetization exchange to acquireinformation regarding the cartilage macromolecular matrix. qMT imagingtechniques typically require multiple MT-contrast images with differentmagnetization preparatory pulses resulting in long scan times which havelimited cartilage assessment to ex-vivo specimens. Cross-relaxationimaging (CRI) is a qMT method which can create three-dimensionalparametric maps of articular cartilage measuring the fraction ofmacromolecular bound protons (f), the exchange rate constant betweenmacromolecular bound protons and free water protons (k), and the T2relaxation time of macromolecular bound protons (T2B) with highresolution and relatively short scan time based upon a limited number ofMT-contrast images. The parameter f provides an indirect measure ofmacromolecular content, while the parameters k, and T2B reflect theefficiency of magnetization exchange between macromolecular boundprotons and free water protons and the spin diffusion between protonsites in macromolecules respectively which may be influenced bymacromolecular organization and ultra-structure

Example 6 Micro Imaging for Non Invasive Monitoring of OsteogenicCells-On-Chip

Microfluidic culture devices are attractive systems to modelphysiological and pathological conditions of tissues and organs.Although these devices allow fluorescent and light microcopy imaging ofcultured cells, one of its current limitations is that various types ofanalyses require sacrificing of the culture. The Inventors havepreviously utilized micro imaging systems to monitor stem celldifferentiation in ex-vivo 3D tissue constructs.

Of interest is utilizing optical imaging to non-invasively monitor stemcell survival and differentiation while cultured in an “organ-on-chip”device. Stiffer membrane and microfluidic environment will promote moreefficient osteogenic differentiation.

To explore this possibility, the organ-on-chip was coated with ECMcrosslinked with UV prior to cell seeding. Then mesenchymal stem cellline overexpressing BMP2 and Luciferase reporter genes were seeded onthe coated organ-on-a-chip (see dimensions and the set up formicrofluidic studies in FIG. 1 and supplemented with osteogenic media.The static cultures were performed using 200 μl media reservoirs thatwere changed every other day. The flow studies were performed using 30μl/h flow of media pulled through using specialized pump (FIG. 1).Micrographs were taken twice a week and survival of the cells wasmonitored using bioluminescent imaging. The media was changed to mediawith Luciferin and imagined using IVIS (Perkin Elmer). The osteogenicdifferentiation after 3 weeks of culture in osteogenic media wasmonitored using florescent probes OsteoSense650 and BoneTag800 that wereintroduced 24 hours before the imaging and were imaged using fluorescentimaging (FLI) and near infrared (NIR) imaging, confocal microscopy andimmunostaining

Example 7 Monitoring Cell Proliferation without Harvest or CultureDisruption

A comparison of chips grown in static culture condition to chips grownunder constant flow of media (30 μl/h) was performed along withevaluation of the effect of the flow on cell survival/proliferation ofcells and the extent of osteogenic differentiation. The microscopicimages (FIG. 1A) show proliferation of the cells under the flowconditions, however it is difficult to quantify the extent ofproliferation using this method without disrupting the cultures.Therefore, the Inventors used cell that express Luciferase reporter geneand the cell proliferation was quantified using bioluminescent imaging(BLI) twice a week (FIG. 2B, C). This imaging method allowed monitor theproliferation of the cells without the need to harvest or disrupt theculture and significant advantage to the flow system was observed. Alsomicrofluidic environment had positive effect on osteogenicdifferentiation, when compared with static cultures.

This effect was observed in fluorescent imaging of osteogenicdifferentiation probes using two different systems—FLI and Near Infrared(FIG. 3). The probes can be detected using different wavelengths offluorescence, therefore both probes can be added simultaneously andimaged separately. The quantification of FLI (FIG. 3A, B) of BoneTagshowed higher osteogenic differentiation of the cell under the flowconditions. OsteoSense was also imaged using confocal microscopy inconjugation with Live/Dead staining (FIG. 3C) showing that most of thelive cells absorbed OsteoSense probe and again the flow chips werestained in more efficiently than the static cultures. The NIR system isconsidered more sensitive and the quantification of the image moreaccurate. Here, the Inventors demonstrate that they system is capable ofdetecting the same trend using both probes (FIG. 3D).

Example 8 Confirmation of Osteogenic Differentiation

In order to confirm osteogenic differentiation of the MSSC-BMP2 cells,the harvested chips were sectioned using vibratome creating transverssections across the channels. Then these sections were subjected toimmunofluorescent staining using primary antibody against Osteocalcinand Bone Sialoprotein (BSP) osteogenic markers. The staining shows cellson both sides of the membrane in both conditions, but mainly in the topchannel. In both conditions there was positive staining for both marker,indicating osteogenic differentiation, however the staining looks moreprominent in the chips that were cultured in flow (FIG. 4 bottom panel).

Organ-on-chip system allows monitoring of the cell survival andproliferation in vitro using BLI imaging system and monitor theosteogenic differentiation of the cell on the chip in real time, withoutthe need of harvesting the cells and disrupting the culture conditions.Here, the Inventors demonstrate that the flow conditions affect bothproliferation and the differentiation of the MSCs that overexpress BMP2.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are sources of mesenchymal stemcells, osteoblasts, bone cells or stem cells, seeding and culturing on amicrofluidic device, imaging methods, including labeling and detection,and the particular use of the products created through the teachings ofthe invention. Various embodiments of the invention can specificallyinclude or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

1-23. (canceled)
 24. A method of culturing mesenchymal stem cells (MSCs)comprising flowing culture media over said MSCs in a microfluidic deviceto produce a higher number of proliferated MSCs in culture than in theabsence of said flowing.
 25. The method of claim 24, wherein themicrofluidic device comprises one or more channels.
 26. The method ofclaim 24, wherein the flow rate of said culture media is 30 ul per hour.27. The method of claim 24, wherein the microfluidic device comprises amembrane and said cells are attached to said membrane.
 28. The method ofclaim 24, wherein proliferation of the cells is monitored withoutdisrupting the culture.
 29. The method of claim 24, wherein the cellsare cultured for three weeks.
 30. The method of claim 24, furthercomprising, adding one or more labeling agents to the microfluidicdevice; and detecting the labeling agent.
 31. The method of claim 24,further comprising, detecting one or more macromolecules secreted by theMSCs.
 32. A method of osteogenic differentiation comprising flowingosteogenic media over mesenchymal stem cells (MSCs) in anorgan-on-a-chip device to produce a higher number of differentiated MSCsin culture that express osteocalcin and bone sialoprotein (bsp)osteogenic markers than in the absence of said flowing.
 33. The methodof claim 32, wherein the microfluidic device comprises one or morechannels.
 34. The method of claim 32, wherein the flow rate of saidculture media is 30 ul per hour.
 35. The method of claim 32, wherein themicrofluidic device comprises a membrane and said cells are attached tosaid membrane.
 36. The method of claim 32, wherein the cells arecultured for three weeks.
 37. The method of claim 32, furthercomprising, adding one or more labeling agents to the microfluidicdevice; and detecting the labeling agent.